Phytoplankton exudates and lysates support distinct microbial consortia with specialized metabolic and ecophysiological traits.

Blooms of marine phytoplankton fix complex pools of dissolved organic matter (DOM) that are thought to be partitioned among hundreds of heterotrophic microbes at the base of the food web. While the relationship between microbial consumers and phytoplankton DOM is a key component of marine carbon cycling, microbial loop metabolism is largely understood from model organisms and substrates. Here, we took an untargeted approach to measure and analyze partitioning of four distinct phytoplankton-derived DOM pools among heterotrophic populations in a natural microbial community using a combination of ecogenomics, stable isotope probing (SIP), and proteomics. Each 13C-labeled exudate or lysate from a diatom or a picocyanobacterium was preferentially assimilated by different heterotrophic taxa with specialized metabolic and physiological adaptations. Bacteroidetes populations, with their unique high-molecular-weight transporters, were superior competitors for DOM derived from diatom cell lysis, rapidly increasing growth rates and ribosomal protein expression to produce new relatively high C:N biomass. Proteobacteria responses varied, with relatively low levels of assimilation by Gammaproteobacteria populations, while copiotrophic Alphaproteobacteria such as the Roseobacter clade, with their diverse array of ABC- and TRAP-type transporters to scavenge monomers and nitrogen-rich metabolites, accounted for nearly all cyanobacteria exudate assimilation and produced new relatively low C:N biomass. Carbon assimilation rates calculated from SIP data show that exudate and lysate from two common marine phytoplankton are being used by taxonomically distinct sets of heterotrophic populations with unique metabolic adaptations, providing a deeper mechanistic understanding of consumer succession and carbon use during marine bloom events.

Recovery and Community Succession of the Zostera marina Rhizobiome after Transplantation.

Seagrasses can form mutualisms with their microbiomes that facilitate the exchange of energy sources, nutrients, and hormones and ultimately impact plant stress resistance. Little is known about community succession within the belowground seagrass microbiome after disturbance and its potential role in the plant's recovery after transplantation. We transplanted Zostera marina shoots with and without an intact rhizosphere and cultivated plants for 4 weeks while characterizing microbiome recovery and effects on plant traits. Rhizosphere and root microbiomes were compositionally distinct, likely representing discrete microbial niches. Furthermore, microbiomes of washed transplants were initially different from those of sod transplants and recovered to resemble an undisturbed state within 14 days. Conspicuously, changes in the microbial communities of washed transplants corresponded with changes in the rhizosphere sediment mass and root biomass, highlighting the strength and responsive nature of the relationship between plants, their microbiome, and the environment. Potential mutualistic microbes that were enriched over time include those that function in the cycling and turnover of sulfur, nitrogen, and plant-derived carbon in the rhizosphere environment. These findings highlight the importance and resilience of the seagrass microbiome after disturbance. Consideration of the microbiome will have meaningful implications for habitat restoration practices.IMPORTANCE Seagrasses are important coastal species that are declining globally, and transplantation can be used to combat these declines. However, the bacterial communities associated with seagrass rhizospheres and roots (the microbiome) are often disturbed or removed completely prior to transplantation. The seagrass microbiome benefits seagrasses through metabolite, nutrient, and phytohormone exchange and contributes to the ecosystem services of seagrass meadows by cycling sulfur, nitrogen, and carbon. This experiment aimed to characterize the importance and resilience of the seagrass belowground microbiome by transplanting Zostera marina with and without intact rhizospheres and tracking microbiome and plant morphological recovery over 4 weeks. We found the seagrass microbiome to be resilient to transplantation disturbance, recovering after 14 days. Additionally, microbiome recovery was linked with seagrass morphology, coinciding with increases in the rhizosphere sediment mass and root biomass. The results of this study can be used to include microbiome responses in informing future restoration work.

Comparative genomic analysis of Vibrios yields insights into genes associated with virulence towards C. gigas larvae.

BACKGROUND: Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae. RESULTS: V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae. CONCLUSIONS: These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

Sipros Ensemble improves database searching and filtering for complex metaproteomics.

Motivation: Complex microbial communities can be characterized by metagenomics and metaproteomics. However, metagenome assemblies often generate enormous, and yet incomplete, protein databases, which undermines the identification of peptides and proteins in metaproteomics. This challenge calls for increased discrimination of true identifications from false identifications by database searching and filtering algorithms in metaproteomics. Results: Sipros Ensemble was developed here for metaproteomics using an ensemble approach. Three diverse scoring functions from MyriMatch, Comet and the original Sipros were incorporated within a single database searching engine. Supervised classification with logistic regression was used to filter database searching results. Benchmarking with soil and marine microbial communities demonstrated a higher number of peptide and protein identifications by Sipros Ensemble than MyriMatch/Percolator, Comet/Percolator, MS-GF+/Percolator, Comet & MyriMatch/iProphet and Comet & MyriMatch & MS-GF+/iProphet. Sipros Ensemble was computationally efficient and scalable on supercomputers. Availability and implementation: Freely available under the GNU GPL license at http://sipros.omicsbio.org. Contact: cpan@utk.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

BACKGROUND: Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. RESULTS: The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. CONCLUSION: Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

Chloroflexi CL500-11 Populations That Predominate Deep-Lake Hypolimnion Bacterioplankton Rely on Nitrogen-Rich Dissolved Organic Matter Metabolism and C1 Compound Oxidation.

The Chloroflexi CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides. In situ expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat.

Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

Introduction: The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination. Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment. Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%. Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Proteogenomic basis for ecological divergence of closely related bacteria in natural acidophilic microbial communities.

Bacterial species concepts are controversial. More widely accepted is the need to understand how differences in gene content and sequence lead to ecological divergence. To address this relationship in ecosystem context, we investigated links between genotype and ecology of two genotypic groups of Leptospirillum group II bacteria in comprehensively characterized, natural acidophilic biofilm communities. These groups share 99.7% 16S rRNA gene sequence identity and 95% average amino acid identity between their orthologs. One genotypic group predominates during early colonization, and the other group typically proliferates in later successional stages, forming distinct patches tens to hundreds of micrometers in diameter. Among early colonizing populations, we observed dominance of five genotypes that differed from each other by the extent of recombination with the late colonizing type. Our analyses suggest that the specific recombinant variant within the early colonizing group is selected for by environmental parameters such as temperature, consistent with recombination as a mechanism for ecological fine tuning. Evolutionary signatures, and strain-resolved expression patterns measured via mass spectrometry-based proteomics, indicate increased cobalamin biosynthesis, (de)methylation, and glycine cleavage in the late colonizer. This may suggest environmental changes within the biofilm during development, accompanied by redirection of compatible solutes from osmoprotectants toward metabolism. Across 27 communities, comparative proteogenomic analyses show that differential regulation of shared genes and expression of a small subset of the approximately 15% of genes unique to each genotype are involved in niche partitioning. In summary, the results show how subtle genetic variations can lead to distinct ecological strategies.

Woronichinia naegeliana: A common nontoxigenic component of temperate freshwater cyanobacterial blooms with 30% of its genome in transposons.

Monitoring in the U.S. state of Washington across the period 2007-2019 showed that Woronichinia has been present in many lakes state-wide. This cyanobacterium was commonly dominant or sub-dominant in cyanobacterial blooms in the wet temperate region west of the Cascade Mountains. In these lakes, Woronichinia often co-existed with Microcystis, Dolichospermum and Aphanizomenon flos-aquae and the cyanotoxin microcystin has often been present in those blooms, although it has not been known whether Woronichinia is a toxin producer. We report the first complete genome of Woronichinia naegeliana WA131, assembled from the metagenome of a sample collected from Wiser Lake, Washington, in 2018. The genome contains no genes for cyanotoxin biosynthesis or taste-and-odor compounds, but there are biosynthetic gene clusters for other bioactive peptides, including anabaenopeptins, cyanopeptolins, microginins and ribosomally produced, post-translationally modified peptides. Genes for photosynthesis, nutrient acquisition, vitamin synthesis and buoyancy that are typical of bloom-forming cyanobacteria are present, although nitrate and nitrite reductase genes are conspicuously absent. However, the 7.9 Mbp genome is 3-4 Mbp larger than those of the above-mentioned frequently co-existing cyanobacteria. The increased genome size is largely due to an extraordinary number of insertion sequence elements (transposons), which account for 30.3% of the genome and many of which are present in multiple copies. The genome contains a relatively large number of pseudogenes, 97% of which are transposase genes. W. naegeliana WA131 thus seems to be able to limit the potentially deleterious effects of high rates of recombination and transposition to the mobilome fraction of its genome.

7-epi-cylindrospermopsin and microcystin producers among diverse Anabaena/Dolichospermum/Aphanizomenon CyanoHABs in Oregon, USA.

Several genomes of Nostocales ADA clade members from the US Pacific Northwest were recently sequenced. Biosynthetic genes for microcystin, cylindrospermopsin or anatoxin-a were present in 7 of the 15 Dolichospermum/Anabaena strains and none of the 5 Aphanizomenon flos-aquae (AFA) strains. Toxin analyses (ELISA and LC-MS/MS) were conducted to quantitate and identify microcystin (MC) and cylindrospermopsin (CYN) congeners/analogs in samples dominated by Dolichospermum spp. of known genome sequence. MC-LR was the main congener produced by Dolichospermum spp. from Junipers Reservoir, Lake Billy Chinook and Odell Lake, while a congener provisionally identified as [Dha7]MC-HtyR was produced by a Dolichospermum sp. in Detroit Reservoir. A second Dolichospermum sp. from Detroit Reservoir was found to produce 7-epi-CYN, with 7-deoxy-CYN also present, but no CYN. The monitoring history of each of these lakes indicates the capacity for high levels of cyanotoxins during periods when Dolichospermum spp. are the dominant cyanobacteria. The diversity of ADA strains found in the US Pacific NW emphasizes the importance of these cyanobacteria as potentially toxic HAB formers in this temperate climatic region. Our results linking congener and genetic identity add data points that will help guide development of improved tools for predicting congener specificity from cyanotoxin gene sequences.

Genome sequence of freshwater nontoxigenic Limnoraphis associated with microcystin-producing blooms.

A sample from a 2019 cyanobacterial bloom in a freshwater reservoir in eastern Oregon, USA, was used to produce a metagenome from which the complete, circular 7.3 Mbp genome of Limnoraphis sp. WC205 was assembled. The Limnoraphis sp. WC205 genome contains gas vesicle genes, genes for N2-fixation and genes for both phycocyanin- and phycoerythrin-containing phycobilisomes. Limnoraphis was present in Willow Creek Reservoir throughout the summer and fall, coexisting with various other cyanobacteria in blooms that were associated with microcystin. The absence of cyanotoxin genes from the Limnoraphis sp. WC205 genome showed this cyanobacterium to be non-toxigenic, although it is predicted to produce cyanobactins closely related to Microcystis aeruginosa microcyclamides. DNA sequence corresponding to the Microcystis mcyG gene identified Microcystis as the microcystin producer in this lake.

Islet autoantibody seroconversion in type-1 diabetes is associated with metagenome-assembled genomes in infant gut microbiomes.

The immune system of some genetically susceptible children can be triggered by certain environmental factors to produce islet autoantibodies (IA) against pancreatic ß cells, which greatly increases their risk for Type-1 diabetes. An environmental factor under active investigation is the gut microbiome due to its important role in immune system education. Here, we study gut metagenomes that are de-novo-assembled in 887 at-risk children in the Environmental Determinants of Diabetes in the Young (TEDDY) project. Our results reveal a small set of core protein families, present in >50% of the subjects, which account for 64% of the sequencing reads. Time-series binning generates 21,536 high-quality metagenome-assembled genomes (MAGs) from 883 species, including 176 species that hitherto have no MAG representation in previous comprehensive human microbiome surveys. IA seroconversion is positively associated with 2373 MAGs and negatively with 1549 MAGs. Comparative genomics analysis identifies lipopolysaccharides biosynthesis in Bacteroides MAGs and sulfate reduction in Anaerostipes MAGs as functional signatures of MAGs with positive IA-association. The functional signatures in the MAGs with negative IA-association include carbohydrate degradation in lactic acid bacteria MAGs and nitrate reduction in Escherichia MAGs. Overall, our results show a distinct set of gut microorganisms associated with IA seroconversion and uncovered the functional genomics signatures of these IA-associated microorganisms.

Revealing General Patterns of Microbiomes That Transcend Systems: Potential and Challenges of Deep Transfer Learning.

A growing body of research has established that the microbiome can mediate the dynamics and functional capacities of diverse biological systems. Yet, we understand little about what governs the response of these microbial communities to host or environmental changes. Most efforts to model microbiomes focus on defining the relationships between the microbiome, host, and environmental features within a specified study system and therefore fail to capture those that may be evident across multiple systems. In parallel with these developments in microbiome research, computer scientists have developed a variety of machine learning tools that can identify subtle, but informative, patterns from complex data. Here, we recommend using deep transfer learning to resolve microbiome patterns that transcend study systems. By leveraging diverse public data sets in an unsupervised way, such models can learn contextual relationships between features and build on those patterns to perform subsequent tasks (e.g., classification) within specific biological contexts.

Predictable Changes in Eelgrass Microbiomes with Increasing Wasting Disease Prevalence across 23° Latitude in the Northeastern Pacific.

Predicting outcomes of marine disease outbreaks presents a challenge in the face of both global and local stressors. Host-associated microbiomes may play important roles in disease dynamics but remain understudied in marine ecosystems. Host-pathogen-microbiome interactions can vary across host ranges, gradients of disease, and temperature; studying these relationships may aid our ability to forecast disease dynamics. Eelgrass, Zostera marina, is impacted by outbreaks of wasting disease caused by the opportunistic pathogen Labyrinthula zosterae. We investigated how Z. marina phyllosphere microbial communities vary with rising wasting disease lesion prevalence and severity relative to plant and meadow characteristics like shoot density, longest leaf length, and temperature across 23° latitude in the Northeastern Pacific. We detected effects of geography (11%) and smaller, but distinct, effects of temperature (30-day max sea surface temperature, 4%) and disease (lesion prevalence, 3%) on microbiome composition. Declines in alpha diversity on asymptomatic tissue occurred with rising wasting disease prevalence within meadows. However, no change in microbiome variability (dispersion) was detected between asymptomatic and symptomatic tissues. Further, we identified members of Cellvibrionaceae, Colwelliaceae, and Granulosicoccaceae on asymptomatic tissue that are predictive of wasting disease prevalence across the geographic range (3,100 kilometers). Functional roles of Colwelliaceae and Granulosicoccaceae are not known. Cellvibrionaceae, degraders of plant cellulose, were also enriched in lesions and adjacent green tissue relative to nonlesioned leaves. Cellvibrionaceae may play important roles in disease progression by degrading host tissues or overwhelming plant immune responses. Thus, inclusion of microbiomes in wasting disease studies may improve our ability to understand variable rates of infection, disease progression, and plant survival. IMPORTANCE The roles of marine microbiomes in disease remain poorly understood due, in part, to the challenging nature of sampling at appropriate spatiotemporal scales and across natural gradients of disease throughout host ranges. This is especially true for marine vascular plants like eelgrass (Zostera marina) that are vital for ecosystem function and biodiversity but are susceptible to rapid decline and die-off from pathogens like eukaryotic slime-mold Labyrinthula zosterae (wasting disease). We link bacterial members of phyllosphere tissues to the prevalence of wasting disease across the broadest geographic range to date for a marine plant microbiome-disease study (3,100 km). We identify Cellvibrionaceae, plant cell wall degraders, enriched (up to 61% relative abundance) within lesion tissue, which suggests this group may be playing important roles in disease progression. These findings suggest inclusion of microbiomes in marine disease studies will improve our ability to predict ecological outcomes of infection across variable landscapes spanning thousands of kilometers.

Proteome changes in the initial bacterial colonist during ecological succession in an acid mine drainage biofilm community.

Proteomes of acid mine drainage biofilms at different stages of ecological succession were examined to understand microbial responses to changing community membership. We evaluated the degree of reproducibility of the community proteomes between samples of the same growth stage and found stable and predictable protein abundance patterns across time and sampling space, allowing for a set of 50 classifier proteins to be identified for use in predicting growth stages of undefined communities. Additionally, physiological changes in the dominant species, Leptospirillum Group II, were analysed as biofilms mature. During early growth stages, this population responds to abiotic stresses related to growth on the acid mine drainage solution. Enzymes involved in protein synthesis, cell division and utilization of 1- and 2-carbon compounds were more abundant in early growth stages, suggesting rapid growth and a reorganization of metabolism during biofilm initiation. As biofilms thicken and diversify, external stresses arise from competition for dwindling resources, which may inhibit cell division of Leptospirillum Group II through the SOS response. This population also represses translation and synthesizes more complex carbohydrates and amino acids in mature biofilms. These findings provide unprecedented insight into the physiological changes that may result from competitive interactions within communities in natural environments.

Ecological distribution and population physiology defined by proteomics in a natural microbial community.

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.

Complete genomes derived by directly sequencing freshwater bloom populations emphasize the significance of the genus level ADA clade within the Nostocales.

The genome sequences of 16 Nostocales cyanobacteria have been determined. Most of them are complete or near-complete genome sequences derived by long-read metagenome sequencing of recent harmful algal blooms (HABs) in freshwater lakes without the potential bias of culture isolation. The genomes are all members of the recently recognized ADA clade (Driscoll et al., Harmful Algae, 77:93, 2018), which we argue represents a genus. We identify 10 putative species-level branches within the clade, on the basis of 91-gene phylogenomic and average nucleotide identity analyses. The assembled genomes each correspond to a single morphotype in the original sample, but distinct genomes from different HABs in some cases correspond to similar morphotypes. We present data indicating that the ADA clade is a highly significant component of current cyanobacterial HABs, including members assigned to the prevalent Dolichospermum and Aphanizomenon genera, as well as Cuspidothrix and Anabaena. In general, currently used genus and species names within the ADA clade are not monophyletic. We infer that the morphological characters routinely used in taxonomic assignments are not reliable for discriminating species within the ADA clade. Taxonomic revisions will be needed to create a genus with a single name (we recommend Anabaena) and to adopt species names that do not depend on morphological traits that lack sufficient discrimination and specificity, while recognizing the utility of some easily observable and distinct morphologies.

Comparative genomics of the ADA clade within the Nostocales.

The ADA clade of Nostocales cyanobacteria, a group that is prominent in current harmful algal bloom events, now includes over 40 genome sequences with the recent addition of sixteen novel sequenced genomes (Dreher et al., Harmful Algae, 2021). Fourteen genomes are complete (closed), enabling highly detailed assessments of gene content and genome architecture. ADA genomes contain 5 rRNA operons, genes expected to support a photoautotrophic and diazotrophic lifestyle, and a varied array of genes for the synthesis of bioactive secondary metabolites. Genes for the production of the taste-and-odor compound geosmin and the four major classes of cyanotoxins - anatoxin-a, cylindrospermopsin, microcystin and saxitoxin - are represented in members of the ADA clade. Notably, the gene array for the synthesis of cylindrospermopsin by Dolichospermum sp. DET69 was located on a plasmid, raising the possibility of facile horizontal transmission. However, genes supporting independent conjugative transfer of this plasmid are lacking. Further, analysis of genomic loci containing this and other cyanotoxin gene arrays shows evidence that these arrays have long-term stability and do not appear to be genomic islands easily capable of horizontal transmission to other cells. There is considerable diversity in the gene complements of individual ADA genomes, including the variable presence of physiologically important genes: genomes in three species-level subclades lack the gas vesicle genes that facilitate a planktonic lifestyle, and, surprisingly, the genome of Cuspidothrix issatschenkoi CHARLIE-1, a reported diazotroph, lacks the genes for nitrogen fixation. Notably, phylogenetically related genomes possess limited synteny, indicating a prominent role for chromosome rearrangements during ADA strain evolution. The genomes contain abundant insertion sequences and repetitive transposase genes, which could be the main drivers of genome rearrangement through active transposition and homologous recombination. No prophages were found, and no evidence of viral infection was observed in the bloom population samples from which the genomes discussed here were derived. Phages thus seem to have a limited influence on ADA evolution.

Computational prediction and experimental validation of signal peptide cleavages in the extracellular proteome of a natural microbial community.

An integrated computational/experimental approach was used to predict and identify signal peptide cleavages among microbial proteins of environmental biofilm communities growing in acid mine drainage (AMD). SignalP-3.0 was employed to computationally query the AMD protein database of >16,000 proteins, which resulted in 1,480 predicted signal peptide cleaved proteins. LC-MS/MS analyses of extracellular (secretome) microbial preparations from different locations and developmental states empirically confirmed 531 of these signal peptide cleaved proteins. The majority of signal-cleavage proteins (58.4%) are annotated to have unknown functions; however, Pfam domain analysis revealed that many may be involved in extracellular functions expected within the AMD system. Examination of the abundances of signal-cleaved proteins across 28 proteomes from biofilms collected over a 4-year period demonstrated a strong correlation with the developmental state of the biofilm. For example, class I cytochromes are abundant in early growth states, whereas cytochrome oxidases from the same organism increase in abundance later in development. These results likely reflect shifts in metabolism that occur as biofilms thicken and communities diversify. In total, these results provide experimental confirmation of proteins that are designed to function in the extreme acidic extracellular environment and will serve as targets for future biochemical analysis.

Nutrient enrichment increases size of Zostera marina shoots and enriches for sulfur and nitrogen cycling bacteria in root-associated microbiomes.

Seagrasses are vital coastal ecosystem engineers, which are mutualistically associated with microbial communities that contribute to the ecosystem services provided by meadows. The seagrass microbiome and sediment microbiota play vital roles in belowground biogeochemical and carbon cycling. These activities are influenced by nutrient, carbon and oxygen availability, all of which are modulated by environmental factors and plant physiology. Seagrass meadows are increasingly threatened by nutrient pollution, and it is unknown how the seagrass microbiome will respond to this stressor. We investigated the effects of fertilization on the physiology, morphology and microbiome of eelgrass (Zostera marina) cultivated over 4 weeks in mesocosms. We analyzed the community structure associated with eelgrass leaf, root and rhizosphere microbiomes, and of communities from water column and bulk sediment using 16S rRNA amplicon sequencing. Fertilization led to a higher number of leaves compared with that of eelgrass kept under ambient conditions. Additionally, fertilization led to enrichment of sulfur and nitrogen bacteria in belowground communities. These results suggest nutrient enrichment can stimulate belowground biogeochemical cycling, potentially exacerbating sulfide toxicity in sediments and decreasing future carbon sequestration stocks.